ABSTRACT To interpret the detailed ultrastructural information of the connectomes in Drosophila and other species, it will be necessary to know the physiological functions of synapses between specific cell types. One key step will be the identification of the neurotransmitter receptors that reside at each site and the connectivity of post-synaptic receptors to specific presynaptic partners. This proposal will combine three cutting-edge technologies to establish methods for tagging neurotransmitter receptors and mapping their subcellular location and synaptic partners. The established connectome of the Drosophila visual system provides an ideal platform for these studies. First, CRISPR-based techniques will be used to insert epitope tags into the genes representing selected neurotransmitter receptors. Second, the newly developed technique of expansion microscopy will be used to localize the tagged receptors at a resolution beyond that of standard light microscopes. Third, stochastic, single-cell labeling will be used to express the tagged receptors at post-synaptic sites within individual neurons. Connectivity to pre-synaptic partners will then be assessed using a previously developed method to sparsely label presynaptic active zones. As a proof of principle, two ionotropic and two G-protein coupled receptors that are expressed in the Drosophila visual system will be tagged. In vitro assays will be used to verify that the tags do not disrupt protein receptor activity in vitro. The tags then will be introduced into the endogenous genes. The activity of the modified proteins will be verified in identified neurons within the visual system that are particularly amenable to functional analyses. The techniques proposed here are scalable and applicable to most neurotransmitter receptors expressed in the fly. With minor modifications, we expect that this approach will be readily applicable to the mouse brain. Mapping neurotransmitter receptors onto the connectome will be an important step forward in our understanding of the brain and its control of complex behavior.